LAPTM4A interacts with hOCT2 and regulates its endocytotic recruitment

Abstract

Human organic cation transporter 2 (hOCT2) is involved in the transport of endogenous and exogenous organic cations mainly in cells of the kidney and the brain. Here, we focus on the regulation of hOCT2 by direct protein-protein interaction. Screening within a mating-based split-ubiquitin-yeast-two-hybrid system (mBSUS) revealed the lysosomal-associated protein transmembrane 4 alpha (LAPTM4A) as a potential interacting protein. Interaction of LAPTM4A and hOCT2 was confirmed by pulldown assays, FRET microscopy analysis and immunofluorescence microscopy. Functionally, overexpression of LAPTM4A significantly decreased ASP(+) uptake in HEK293 cells stably transfected with hOCT2, suggesting a negative regulation of hOCT2-mediated transport. Furthermore, overexpression of LAPTM4A leads to a significantly decreased hOCT2 plasma membrane expression in surface biotinylation experiments. In addition, significant expression of LAPTM4A in human kidney was demonstrated by immunoblotting and immunofluorescence.In this work, LAPTM4A has been identified as interaction partner of hOCT2. LAPTM4A regulates the function of hOCT2 by influencing its trafficking to/from the cell membrane and processing it via the intracellular sorting machinery.

CRISPR Screen Reveals that EHEC’s T3SS and Shiga Toxin Rely on Shared Host Factors for Infection

“…A subset of fluorescence emanated from nucleoli, potentially resulting from nonspecific primary antibody binding, although Stx has been reported to be actively transported into nucleoli ( 51 ). Unexpectedly, as prior studies localized LAPTM4A to lysosomes ( 31 – 34 ), we found that a LAPTM4A-GFP fusion protein localized to the Golgi complex, like TM9SF2 ( Fig. 5A ).…”

“…The LAPTM4A locus has been reported to encode a transmembrane protein localized to lysosomes and late endosomes. It has been linked to intracellular transport of nucleosides, multidrug resistance, and maintenance of lysosomal integrity ( 31 – 34 ). In addition to the enrichment of guides targeting TM9SF2, LAPTM4A, and genes related to sphingolipid biosynthesis, our analysis detected enrichment for guides targeting several genes associated with cancer and cell proliferation (MLLT3, TFAP4, ZNF217, and DUSP6) ( 35 – 38 ).…”

Tetraspanin CD63 controls basolateral sorting of organic cation transporter 2 in renal proximal tubules

“…B1–B2‐linked full‐length cDNA for hOCT1, −2, or −3 was cloned in vivo in frame into the expression vector pMetYCgate containing the Cub and the transcription factor protA‐LexA‐VP16 peptide and expressed in the reporter yeast strain THY.AP4. Expression was verified by Western blot analyses (29, 30). Yeast cells expressing hOCTs were cotrans‐formed with a human cDNA kidney library expressed in the vector pPR3‐N (Dualsystems Biotech, Schlieren, Switzerland) along with the Nub.…”

The Role of Transporters in the Toxicity of Chemotherapeutic Drugs: Focus on Transporters for Organic Cations

“…However, it should be considered that the function of these transporters is strongly determined by the interaction with other cellular proteins, which modulate their activity and determine their cellular fate. For example, a direct interaction of hOCT2 with the lysosomal‐associated protein transmembrane 4α (LAPTM4A) has been demonstrated . This interaction influences transporter trafficking to and from the cell membrane and processing via the intracellular sorting machinery.…”

“…For example, a direct interaction of hOCT2 with the lysosomal-associated protein transmembrane 4α (LAPTM4A) has been demonstrated. 138 This interaction influences transporter trafficking to and from the cell membrane and processing via the intracellular sorting machinery. Of course SNPs of proteins that are part of the proteome of the transporters have potentially important implications for transporter function and should be taken into account in future studies.…”